Support materials for high productivity liquid chromatography must be chemically and mechanically stable. Rigid inert polymers such as crosslinked polystyrene permit increases in operating pressure and flow rates. Unfortunately, hydrophobic interactions between such resins and proteins often are so strong that the proteins are denatured upon adsorption or during elution. Thus, it is recognized that one must post treat the hydrophobic surface of such polymeric chromatography-packing materials to adapt it for use in the separation of proteins.
It is often necessary to force liquid through chromatographic column beds because the liquid mobile phase flows too slowly under the force of gravity to produce acceptable separation times. The problem of flow rate is aggravated by the use of microparticulate packing geometries often used to enhance resolution. It is common in liquid chromatography, i.e., high pressure liquid chromatography or HPLC, to use pressures of 100 atmospheres or more to achieve rapid separations. Soft-gel packing materials will not tolerate more than a few atmospheres of pressure, thus are unsuited for high pressure use, and increasingly are being replaced with rigid materials.
It is at least theoretically possible to react hydrophilic moieties with sites on hydrophobic polymers to provide a hydrophilic surface character. Such post coated materials, often referred to as pellicular materials or supports, can be produced readily if the base polymer is one having many easily derivatized reactive groups. One example of this approach involves alkylation of styrene, followed by polymerization to produce derivatized polystyrene, and subsequent surface reaction with polyoxyethylene.
Another example is the derivatization of polymethacrylate. The basic hydrophobic character of this polymer may be altered if very hydrophilic groups are introduced into the surface through esterification. Such hydrophilic groups typically comprise hydroxyl and/or ether groups, and include such materials as glycerol, ethylene glycol, diethylamino ethanol, trimethyl ethanolamino glycolic acid, and hydroxyethylsulfonic acid. Unfortunately, exposure of such materials to extremes of pH, often required for regeneration of a chromatography medium, has the effect of hydrolyzing the ester linkages and of degrading the properties of the medium.
Where the surface of rigid, inert, hydrophobic material has groups reactive enough to permit such derivatization, that property generally is inconsistent with a major reason for turning to such support materials in the first place, i.e., inertness. However, the art has developed strageties to provide coatings on truly inert polymeric particles without requiring formation of covalent bonds directly between the coating and the support.
An example of alteration of the surface characteristics of a hydrophobic support medium involves adsorbing a water soluble surfactant onto a silica-based reverse-phase packing material. (see Chang et al, J. Chrom, V. 319, pp. 396-399, 1985). The surfactants have long hydrophobic tails and bulky hydrophilic heads. Once the tails are adsorbed to the support, the hydrophilic heads cover the hydrophobic surface so a protein cannot bind. Small molecules can penetrate into the hydrophobic region, and the adsorbed coating is stable only in polar solvents. Further, the surfactants can leach from the sorbent surface, and very hydrophobic proteins can displace surfactant molecules, thereby degrading the support.
U.S. Pat. No. 4,245,005 describes an elegant method for producing a pellicular polyethyleneimine-coated, porous support. Charged polymeric molecules are attracted to the surface of support materials of opposite charge by means of electrostatic forces. Once adsorbed, the polymeric materials are cross-linked in place and therefore resist erosion by solvent extraction, changes in pH, or exposure to elution buffers. This technique works well for the production of cationic resins (exchange anions) but is impractical for the preparation of organic, chromatographic packing materials useful in other types of chromatography procedures.